PROJECT SUMMARY/ABSTRACT We know surprisingly little about the roles of individual caspases in cell death despite members of this protease family having been associated with apoptosis for over two decades. The current compendium of caspase peptide inhibitors and substrates are highly promiscuous and do not provide the resolution required to assign any current cell-based observations to individual caspases, whereas genetically engineered systems offer tenuous clues to caspase function but only in somewhat artificial settings. For example, the highly homologous executioner caspases-3 and -7 that are assumed to target conserved biological substrates during the final stages of apoptosis are indistinguishable with respect to their canonical recognition peptide sequence, DEVD. However, knockout mouse models of either caspase-3 or -7 harbor divergent phenotypes and studies employing cell-free lysates indicate these two caspases may have distinct cellular targets. Thus, there is an urgent need to decipher the roles of specific caspases within cells and to control their activities. Here, our goal is to develop novel peptide-based probes, inhibitors, and substrates capable of specific detection and regulation of caspase-3 and caspase-7. Our technological advances will enable us to provide an unprecedented evaluation of the biological activities of each individual caspase during apoptosis at resolutions not previously possible. We have amassed substantial preliminary data employing our new methodology incorporating unnatural amino acids and novel warheads to identify first-in-class cell-permeable peptide-based inhibitors and substrates for caspase-3 with 100x selectivity over caspase-7 and all other caspases. We will continue to develop exquisitely selective peptides as covalent activity-based probes that target either caspase- 3 or -7 and these peptides will be rapidly converted into highly specific inhibitors and FRET-based substrates for cell studies. We will then be able to deconvolute the specific contributions and substrates of caspases-3 and -7 during apoptosis with kinetic precision. We also propose to identify and characterize small molecule activators of the inactive pro-form of each caspase so that each protease can be selectively activated within cells and establish these proteases as bona fide drug discovery targets. Our high-throughput FP-based assay against wild-type procaspases has already identified robust caspase activators and our expertise in validation of such compounds has been demonstrated with proof-of-concept procaspase-3 activators. Establishing caspase activator specificity in cells has been exceeding difficult, as caspases-3 and -7 are eventually turned on by all cell death initiators. However, our new probes and insights on specific caspase-3 and -7 cellular substrates will be indispensable for compound validation during activator-induced apoptosis. This cutting edge project on identifying the role of individual caspases in apoptosis will be of significant value to the general research community and can be extrapolated to include other biological processes and human diseases implicated with caspases, including cellular differentiation, neurological diseases, and ischemia.